Anti-bacterial compositions

ABSTRACT

Anti-bacterial composition comprising an admixture of an organic acid (excluding acetate, propionate and butyrate) together with a coumarin or coumarin glycoside. Preferred organic acids include lactate, citrate and benzoate, especially L-lactate. Preferred coumarins are esculetin, scopoletin, imbelliferone and Coumarin (1,2-benzopyrone). The composition, which is effective against  E. coli  0157,  Salmonella, Listeria, Campylobacter  and MRSA, can be used to disinfect buildings or instruments and in food preparation, e.g., as a vegetable wash.

The present invention relates to food safety and also to the treatmentof bacterial infections, in particular due to E. coli O157 and otherfoodborne pathogenic bacteria.

Foodborne bacterial pathogens are a major cause of concern to publichealth, presenting the food industry with a severe challenge.Escherichia coli O157 is a prime example. This bacterium is thecausative agent of haemorrhagic colitis and haemolytic uremic syndrome.Infections caused by E. coli O157, though infrequent, are associatedwith a high level of morbidity and mortality, particularly in the youngand the elderly. The severity of infections caused by E. coli O157 andother pathogenic bacteria has attracted a high level of media attention.This has resulted in reduced public confidence in food safety,particularly in red meat products and products such as salad leaves andother vegetables which may be treated with fertilisers containing animalmanure.

There is a growing interest in how bacterial pathogens enter the foodchain and practical measures of prevention. Escherichia coli O157, forexample, has a very low infectious dose and it may be carriedasymptomatically by farm animals including cattle, sheep, pigs, turkeysetc. Farm animals including cattle and sheep are regarded as a primaryreservoir of E. coli O157. Moreover, this organism is repeatedlyisolated from the farmyard which strongly implicates this environment inthe persistence of E. coli O157 (LeJeune et al. (2001)). Cattle faecesare a major source of contamination of meat products in slaughterhouses, and slurry is a potential source of contamination of local watersupplies and crops, as well as vegetables and fruit which maybe eatenraw. Farm workers and visitors, veterinarians and slaughterhouse staffare also at risk of infection from farm animal faeces. The practice offeed withdrawal during transport to slaughter was introduced as ameasure to control the amount of faecal contamination on hides (Leitchet al. (2001)). However, feed deprivation is thought to predisposecattle to carriage of E. coli and Salmonella (Brownlie and Grau (1967)).

There is thus a continuing need for new and improved compositionscapable of reducing the numbers of, or inactivating, bacterial pathogens(including those present at any point in the food chain, or present inthe environment). This includes the processes involved in thepreparation of meat and vegetable products for the consumer, and thecontrol of bacterial pathogens present within animals which carry andshed micro-organisms (including, for example, humans, cattle, pigs,sheep, chickens, and turkeys). It also includes the prevention ofproliferation or the reduction in survival of pathogens in animal feedsand the decontamination of water troughs through which infectious agentsmay spread from animal to animal or from animal to human. It especiallyincludes applications to reduce the presence of pathogenic bacteria onthe surfaces of fruits and vegetables and on surfaces (for examplefloors, benches, work tops, walls, cutting implements), or present onthe surface of fish, shellfish, raw cut meat products and animalcarcasses.

With regard to reducing or eliminating bacterial pathogens in thepreparation of meat and vegetable products for the consumer, the variouspotential control stages in the food chain are referred to as HazardAnalysis Critical Control Points (HACCP). E. coli O157 is an importantpathogen and is thus of particular interest, but other bacterialpathogens which may be carried and shed from host animals include, forexample, Salmonella species (including S. enteritidis), Campylobacterspp., Staphylococcus spp., Listeria monocytogenes as well as non-O157 E.coli strains causing food-borne infections.

The technology developed to control food borne pathogens could also findapplication for other bacterial pathogens. Compounds or proceseseffective against the range of bacterial pathogens mentioned above wouldbe likely to be active against other bacterial pathogens found inhabitats other than food. An example is the control ofmethicillin-resistant Staphylococcus aureus (MRSA). This bacterium canbe found on unprotected surfaces in hospitals (floors and walls,implements etc) and novel disinfectants could be invaluable in itscontrol.

Currently there are limited and relatively inefficient means ofcontrolling the contamination of the food chain and the environment byE. coli O157 and other pathogenic bacteria, treating human infection,and reducing carriage of pathogenic bacteria by animals such as cattle,sheep, and humans. Indeed, certain antibiotic therapies for treatment ofhumans infected with E. coli O157 causes lysis of the bacterium andsubsequent release of the potent disease-causing toxin.

Current methods of reducing and killing bacterial pathogens on surfacessuch as abattoir and butchers' floors and work benches comprise washingand soaking such surfaces in chlorinated solutions. However, reportshave suggested that chlorine does not effectively kill E. coli O157.Furthermore, resistance to this chemical may occur (see Beuchat (1999)Journal of Food Protection 62(8): 845-849; Cutter et al. (1995) Journalof Food Safety 15(1): 67-75; Lisle et al. (1998) Applied andEnvironmental Microbiology 64(12): 4658-4662; and Zhao et al. (2001)Journal of Food Protection 64(10): 1607-1609). In addition, chlorine canalter the taste and smell of foods in contact with the surface, reducingpalatability. These disadvantages are also inherent in the use ofchlorinated water for the washing of fruits, vegetable leaves andsimilar materials.

There are a number of publications describing the inhibition ofbacterial pathogens on fresh food items. WO-A-99/44444 describes the useof a solution containing lactic acid and at least one ingredient chosenfrom hydrogen peroxide, sodium benzoate or glycerolmonolaurate atcertain temperatures and durations. No mention is made of thedistinction between the L- and D-isomers of lactic acid, nor is thereany mention of coumarin compounds.

Castillo et al. (2001) describe the use of L-lactate in reducing thepresence of pathogenic bacteria on hot beef carcasses and suggest thatthe technology may be useful at lower temperatures. No mention is madeas to an additive effect of L-lactate with coumarin compounds, nor isthere any mention as to the use of L-lactate in reducing, inhibiting orkilling bacteria in vivo in animals or humans. Certain companies andresearchers have published the use of Lactobacillus species (NutraceutixInc.) and certain E. coli species (U.S. Pat. No. 5,965,128) ininhibiting E. coli O157 in vitro and in vivo. Duncan (1998) describesthe inhibitory effect of esculetin, a coumarin compound, in combinationwith the volatile fatty acids (VFA's) acetate, butyrate and propionatetowards E. coli O157. There is no mention of other organic acids, suchas lactic acid, having any additive or synergistic effect on theinhibitory and killing of E. coli O157. In addition, the most active VFAis butyrate which has an associated undesirable smell rendering thisacid unsuitable for use in the food industry at the concentrationsrequired.

Bintsis et al. (2000) show the efficacy of furocoumarins in inhibitingcertain pathogens, including E. coli O157. However, there is no mentionof the combination of coumarins with lactic acid or other organic acids.

We have now found that the L-isomer of lactate is unexpectedly moreactive than D-lactate against pathogenic E. coli strains (including E.coli O157). Further, we have found that combination of an organic acidwith a coumarin or a glycoside thereof results in a synergisticallyenhanced anti-bacterial effect. The “organic acid” as herein defined maybe any organic acid but specifically excludes the volatile fatty acidsacetate, propionate and butyrate. In one embodiment the organic acid maybe any organic acid but excludes short chain volatile fatty acids.Desirably, the organic acids selected are medium chain fatty acids (eg.heptanoic and decanoic acids), long chain fatty acids (eg. dodecanoicacid), unsaturated acids (eg. sorbic acid), hydroxylic acids (eg. lacticacid and citric acid), aromatic acids (eg. benzoic acid and salicyclicacid) or multicarboxylic acids (eg. citric acid and succinic acid).Preferably the organic acids are hydroxylic acids such as citrate orlactate. L-isomers of optically active hydroxylic acids are preferred.It should be noted that the nomenclature for fatty acids adopted is asset out by Cherrington et al. (1991).

The present invention provides an anti-bacterial composition comprisingan admixture of an organic acid as defined above and a coumarin or acoumarin glycoside. Lactate, citrate and benzoic acid are preferred,especially L-lactate.

The present invention also provides a method for reducing the infectiveability or inactivating bacterial pathogens by contacting said bacteriawith a mixture of an organic acid as defined above and a coumarin or acoumarin glycoside. Further, the method and composition of the presentinvention may be used to reduce the shedding of pathogens (including butnot limited to E. coli O157, Salmonella, Listeria, Campylobacter andMRSA) from animals and humans.

We believe that any coumarin will be effective in the compositiondescribed above. The preferred coumarins are esculetin, scopoletin,umbelliferone and Coumarin, but other coumarins may also be used. Thecoumarins are derivatives of benzo-α-pyrone and occur in plants in thefree state and as glycosides. For clarity, the term “coumarin” will beused to describe the generic group of benzo-α-pyrone compounds, whereas“Coumarin” will refer to 1,2 benzopyrone. In addition to the free statecoumarins, the coumarin glycosides may also be used since these arecommonly converted to the free coumarin in vivo and may have theadvantage of increased solubility which aids administration andabsorbance. Additionally, the sugar moiety, once hydrolysed from theglycoside, can provide a sugar source for growth of beneficial bacteria.An example of a coumarin glycoside is esculin, which is a glycoside ofesculetin. Furocoumarins, for example psoralen, 3-methoxy-psolaren and8-methoxy-psoralen may also be of interest.

Preferably, the composition contains from 1 mM to 500 mM, morepreferably from 20 mM or 50 mM to 250 mM, of organic acid. Preferredorganic acids are lactate, citrate and benzoic acid.

Preferably, the composition contains from 0.05 mM to 15 mM of a coumarinor a coumarin glycoside, preferably at least 0.5 mM, for example 0.68mM, of a coumarin or a coumarin glycoside.

The composition of the present invention has the advantage of beingapplicable to all stages of potential contamination andcross-contamination from farm to fork. Exemplary stages include use inthe treatment of salad leaves and other vegetable material, in animalfeeds, in water troughs on farms, in food preparation from slaughter tosale to the public, for use with plant matter intended as a foodstuffand also for treatment of animals or humans infected with saidpathogens. Further, the composition could be used as a disinfectant,cleaner, sterilizer for commercial and non-commercial cooling devicessuch as fridges and freezers. In particular, the composition could beused to disinfect buildings, in particular public buildings such asschools or hospitals, or to disinfect surfaces (such as floor, walls,furniture and medical devices/implements). The composition could furtherbe used in packaging material for foods (such as plastic “clingfilm”wrap) and, since the composition increases in effectiveness withincreasing temperature, would protect wrapped produce taken from therefrigerated conditions in retail premises during transport. Thecomposition could also find use in washing, coating or beingincorporated into bandages, dressings and other coverings used toprotect wounds from infection and contamination.

One of the benefits of the composition of the present invention resideswith the antioxidant properties of coumarins. Antioxidants have wellknown health enhancing and disease preventing properties by virtue inpart to their ability to reduce oxidative damage to our cells which maylead to development of many clinical conditions including cancers, heartdisease, Alzheimer's disease and arthritis. Accordingly, in addition tothe ability of coumarins to inhibit and kill pathogens, ingestion byanimals and humans will enhance the health status of the recipient andmay help to prevent and treat diseases related to oxidative stress.Apart from the possible benefits to health outlined above, theantioxidant properties of the coumarins are likely to extend theshelf-life of treated food or feed stuffs.

The composition of the present invention may be administered to animalsto reduce shedding of the pathogens into the environment, for examplefields, farms, water supplies, slurry and vehicles used fortransportation of animals. Reduction of shedding leads to a lowering ofthe risk of contamination of the environment and edible and non-edibleagricultural products such as fruit and vegetables and will also reducethe risk of infection to other animals and humans.

Preferably the method of the present invention is conducted at atemperature of 37° C. or lower, for example 20° C. or 5° C.(representing body temperature, ambient temperature and refrigeratedtemperature respectively). In general, increasing the temperatureincreases the effectiveness of the composition. However, even at 5° C.,the numbers of Salmonella, for example, are reduced by greater than 5log₁₀ units within 60 minutes. At 20° C., a greater than 5 log₁₀ unitdecrease occurs in 5 minutes.

Further the present invention may be administered to humans infectedwith pathogens so as to reduce the numbers, inhibit and kill thepathogens and consequently treat diseases caused by the infectingorganism. For example the composition of the invention could be used totreat humans infected with E. coli O157 or MRSA and thus prevent orameliorate the effects of infection with such pathogens, which includehaemorrhagic colitis and haemolytic uremic syndrome and sepicaemia.

In one embodiment, the composition of the present invention may furthercomprise a volatile fatty acid (VFA) such as, for example, acetate,butyrate or propionate. Alternatively or additionally the compositionmay comprise a polyasaccharide or other readily fermentable compoundwhich, upon digestion in the gut, is converted to an acid such aslactate.

The composition can be used in various formats for example as sprays,liquid solutions, gels, packaging and wrapping material for foods foruse on surfaces and can be delivered to the site of action in the rumenor gastro-intestinal tract by oral administration in any appropriatecarrier, excipient, diluent or stabilizer. Such delivery mechanisms maybe of any formulation including but not limited to solid formulationssuch as tablets or capsules or as feed additives; liquid solutions suchas yoghurt or drinks or suspensions.

It is believed that any bacterial pathogen will be adversely affected bythe composition of the present invention. Pathogenic E. coli strains,including E. coli O157, are of particular interest, as are Salmonellaspp, Listeria spp. and Staphylococcus spp., especially MRSA.

The present invention will now be further described with reference tothe following, non-limiting, examples and figures in which:

FIG. 1 shows the susceptibility of E. coli O157:H7 strain NCTC 12900 toL-lactate and D-lactate. The L-lactate solution concentrations were 50,100, 150 and 200 mM and those of D-lactate 100, 150 and 200 mM. Thesolutions all had a final pH of 3.8 and were incubated at 37° C. Thelimit of detection is 50 cfu/ml or 1.7 log₁₀ cfu/ml.

FIG. 2 illustrates the susceptibility of E. coli O157:H7 strain NCTC12900 to various proportions of L-lactate and D-lactate. The solutionconcentrations were 100 mM D-lactate, 75 mM D-lactate+25 mM L-lactate,50 mM D-lactate+50 mM L-lactate, 25 mM D-lactate+75 mM L-lactate, and100 mM L-lactate. The solutions all had a final pH of 3.8 and wereincubated at 37° C. The limit of detection is 50 cfu/ml or 1.7 log₁₀cfu/ml.

FIG. 3 indicates the survival of 8 E. coli O157:H7 strains and 8non-O157 E. coli strains following treatment with 100 mM L-lactate orD-lactate for 3 hours. The solutions all had a final pH of 3.8 and wereincubated at 37° C. The limit of detection is 50 cfu/ml or 1.7 log₁₀cfu/ml.

-   -   Dark Shading=D-Lactate.    -   Light Shading=L-Lactate.

FIG. 4 illustrates the synergy between L-lactate (50 mM) and esculetin(7.5 mM) in reducing the numbers of the E. coli O157:H7 strain NCTC12900. The solutions all had a final pH of 3.8, were incubated at 37° C.and the limit of detection was 50 cfu/ml or 1.7 log₁₀ cfu/ml.

FIG. 5 illustrates the effect of temperature on the antimicrobialefficacy of 200 mM L-lactate and 7.5 mM esculetin on the E. coli 0157:H7strain NCTC 12900. Cultures were incubated at 5, 20 and 37° C. Thesolutions all had a final pH of 3.8 and the limit of detection was 50cfu/ml or 1.7 log₁₀ cfu/ml.

FIGS. 6A-C depict the synergism between various coumarins (7.5 mM) andL-lactate (50 mM) against the E. coli 0157:H7 strain NCTC 12900. Thecoumarins tested were scopoletin (A), Coumarin (B) and umbelliferone(C). Cultures were incubated at 37° C., had a final pH of 3.8 and thelimit of detection was 50 cfu/ml or 1.7 log₁₀ cfu/ml.

FIGS. 7A-B illustrate the synergy between esculetin and 50 mM citricacid (A) or 25 mM benzoic acid in reducing the numbers of the E. coli0157:H7 strain NCTC 12900. The solutions all contained 7.5 mM esculetinand had a final pH of 3.8. The limit of detection was 50 cfu/ml or 1.7log₁₀ cfu/ml.

FIGS. 8A-C indicates the synergy between Coumarin and L-lactate for S.enteritidis (A), L-monocytogenes (B), and an MRSA strain of S. aureus(C). The solutions all contained 10 mM Coumarin and had a final pH of3.8. The limit of detection was 50 cfu/ml or 1.7 log₁₀ cfu/ml.

FIGS. 9A-B compare the effect of temperature on survival of S.enteritidis (A) and L-monocytogenes (B) in L-lactate and Coumarin. Thesolutions contained 10 mM Coumarin and 25 mM L-lactate (A) or 50 mML-lactate (B). The final pH was 3.8 and the limit of detection 50 cfu/mlor 1.7 log₁₀ cfu/ml.

FIG. 10 illustrates the synergy between 2% L-lactate and 6.8 mM Coumarinin reducing the viability of 8 E. coli O157:H7 strains and 8 non-O157:H7E. coli strains determined over 1 hour. Results are shown as percentagesurvival.

FIGS. 11A-D show the synergistic antimicrobial effect of 2% L-lactateand 6.8 mM Coumarin on the E. coli 0157 strain NCTC 12900 (A), S.enteritidis (B), L-monocytogenes (C) and S. aureus (D). The limit ofdetection was 50 cfu/ml or 1.7 log₁₀ cfu/ml.

FIGS. 12A-D illustrate the effect of temperature on the antimicrobialefficacy of 2% L-lactate and 6.8 mM Coumarin for the E. coli 0157:H7strain NCTC 12900 (A), S. enteritidis (B), L. monocytogenes (C) and S.aureus (D). The limit of detection was 50 cfu/ml or 1.7 log₁₀ cfu/ml.

FIGS. 13A-E demonstrate the synergistic growth inhibition of L-lactateand esculetin (A) or L-lactate and Coumarin (B-E) on E. coli O157:H7strain NCTC 12900 (A-B), S. enteritidis (C), L. monocytogenes (D) and S.aureus (E).

EXAMPLES

Table 1 shows the bacterial strains used in the examples. TABLE 1 E.coli Strains Other Strains Strain Origin Serotype Species Strain NCTCHuman O157:H7 Salmonella NCTC 12900 enteritidis 4444 NCTC Human O157:H7Listeria NCTC 13126 monocytogenes 11994 NCTC Human O157:H7Staphylococcus NCTC 12079 aureus 10442 AUIO-5 Cattle O157:H7 faecesAUIO-7 Raw milk O157:H7 AUIO-13 Minced O157:H7 beef AUIO-309 CheeseO157:H7 AUIO-ND Sheep O157:H7 faeces F318 Sheep O162 rumen F38 Sheep Orough rumen EC17 Pig O106:NM EC30 Bison O113:H21 EC33 Sheep O7:H21 EC45Pig ON:HM EC47 Sheep ON:H18 EC67 Goat O4:H43Methods and Results

As the infectious dose of E. coli O157:H7 is very low, our main aim wasto develop a treatment capable of killing high levels of E. coli cells.The E. coli strains were cultured such that there was a population ofapproximately 10⁹ cfu/ml. The final pH for each culture was 3.8, exceptwhere 2% lactate was present in which case the final pH was around 2.0.Treatments consisted of various concentrations of the organic acidsL-lactate, D-lactate, citrate or benzoate and the coumarins esculetin,Coumarin, scopoletin or umbelliferone. These were added to cultureswhich were then incubated at 5° C., 20° C. or 37° C. Samples wereextracted at various time intervals and the population of E. coliO157:H7 was determined. Other pathogens were tested in a similar mannerwith the exception of L. monocytogenes where the starting population was10⁸ cfu/ml.

L-lactate (final concentrations of 50, 100, 150 or 200 mM) or D-lactate(final concentrations of 100, 150 or 200 mM) were added to preparedcultures and incubated at 37° C. As illustrated in FIG. 1, L-lactate wasmore effective at reducing numbers of the E. coli O157:H7 strain NCTC12900 than D-lactate at all concentrations measured. The inactivation ofthis organism by either treatment was dose-dependent.

L-lactate and D-lactate (final concentrations of 100 mM D-lactate, 75 mMD-lactate+25 mM L-lactate, 50 mM D-lactate+50 mM L-lactate, 25 mMD-lactate+75 mM L-lactate, and 100 mM L-lactate) were added to preparedcultures and incubated at 37° C. As shown in FIG. 2, 100 mM L-lactateexerted a greater antimicrobial effect than 100 mM D-lactate on E. coli0157:H7 strain NCTC 12900. Increasing the proportion of the L-isomerover the D-isomer increased the antimicrobial efficacy in adose-dependent manner for both strains.

Treatments consisting of 100 mM of L-lactate or D-lactate were testedagainst 8 E. coli O157:H7 strains and 8 non-O157:H7 E. coli strains. Theviability was determined initially and after 3 hours at 37° C. Thepercentage survival was calculated by dividing the final viability bythe initial viability and multiplying the result by one hundred. Asshown in FIG. 3, the inactivation caused by L-lactate was much greaterthan that of D-lactate for all the E. coli strains tested. This stronglysuggests that the greater susceptibility to L-lactate compared toD-lactate is widespread in E. coli.

The effect of L-lactate (50 mM) and esculetin (7.5 mM) on strain NCTC12900 at 37° C. was determined and the results are shown in FIG. 4. Incombination, these compounds synergistically reduced the survival ofstrain NCTC 12900 by approximately 7 log₁₀ units/ml in 8 hours. Thecombined treatment of L-lactate lactate and esculetin had a greatereffect than the individual treatments, illustrating synergy between thetwo compounds.

The effect of temperature on the antimicrobial efficacy of 200 mML-lactate+7.5 mM esculetin on the E. coli O157:H7 strain NCTC 12900 wastested. The temperatures assayed were 5° C., 20° C. and 37° C. As shownin FIG. 5, as the temperature increased, the efficacy of theantimicrobial compounds increased.

The range of coumarins tested was extended to include scopoletin,Coumarin and umbelliferone and their potential synergy with L-lactateagainst E. coli O157:H7 strain NCTC 12900 was evaluated. As shown inFIG. 6A, neither L-lactate (50 mM) nor scopoletin (7.5 mM) on their ownaffected the viability of this bacterial strain. In combination thesecompounds reduced the numbers of NCTC 12900 by greater than 7 log₁₀units in 8 hours Similarly, neither 7.5 mM Coumarin (FIG. 6B) nor 7.5 mMumbelliferone (FIG. 6C) affected viability, but in concert withL-lactate, these compounds caused a synergistic reduction in viable E.coli O157 cells of greater than 7 log₁₀ units in 6 hours. This stronglysuggests that coumarins in general exhibit an antimicrobial synergy withL-lactate for E. coli strains.

The range of organic acids tested was extended to include citrate andbenzoate and their potential synergy with 7.5 mM esculetin against NCTC12900 evaluated. As shown in FIG. 7A, neither citrate (50 mM) noresculetin (7.5 mM) when used individually affected the viability of theE. coli O157:H7 strain. In combination these compounds reduced thenumbers of NCTC 12900 by approximately 3 log₁₀ units in 8 hours. Asshown in FIG. 7B, benzoate (25 mM) on its own reduced viability byapproximately 4 log₁₀ units in 8 hours. Benzoate with esculetin (7.5 mM)caused a synergistic reduction in viable E. coli O157 cells of greaterthan 7 log₁₀ units in 8 hours. This strongly suggests that organic acidsin general exhibit an antimicrobial synergy with coumarins for E. colistrains.

The antimicrobial effect of L-lactate and Coumarin on pathogens otherthan E. coli was examined. The strains examined were Salmonellaenteritidis NCTC 4444, Listeria monocytogenes NCTC 11994 and themethicillin-resistant Staphylococcus aureus (MRSA) strain NCTC 10442.

As shown in FIG. 8A, Coumarin (10 mM) alone did not affect the viabilityof S. enteritidis whereas L-lactate (25 mM) reduced viability by greaterthan 5 log₁₀ units in 8 hours. Together, Coumarin and L-lactatesynergistically reduced the viability of this organism by greater than 7log₁₀ units in 2 hours.

FIG. 8B illustrates the affect of Coumarin and L-lactate on L.monocytogenes. Coumarin (10 mM) alone had no effect on viability whereasL-lactate (50 mM) reduced viability by greater than 5 log₁₀ units in 8hours. Coumarin and L-lactate together synergistically reduced theviability of this L. monocytogenes strain by greater than 6 log₁₀ unitsin 2 hours.

The viability of S. aureus (FIG. 8C) was not affected by 10 mM Coumarinalone whereas 50 mM L-lactate reduced viability by greater than 3 log₁₀units in 8 hours. The viability of this MRSA strain was reduced byapproximately 6 log₁₀ units in 8 hours when both L-lactate and Coumarinwere present. These results strongly suggest that organic acids andcoumarins exhibit an antimicrobial synergy for bacterial pathogens ingeneral.

The effect of temperature on the antimicrobial efficacy of L-lactate andCoumarin was examined for the S. enteritidis strain (FIG. 9A) and the L.monocytogenes strain (FIG. 9B). For both of these organisms theantimicrobial effect was greater as the temperature increased.

For certain applications it was desirable to determine potential synergybetween L-lactate and Coumarin at the concentrations of these compoundslikely to be used in commercial-like environments. The concentration ofL-lactate was 2% and that of Coumarin 6.8 mM. As shown in FIG. 10, arange of 8 E. coli O157 strains and 8 non-O157 E. coli strains weretested against these compounds and the viability of the strainsdetermined at the start of the experiment and after 1 hour. Survival wascalculated as previously. Coumarin alone had little effect on thesurvival of the E. coli strains whereas L-lactate reduced survival tobetween 0.00001% and 46%. L-lactate and Coumarin in combination reducedsurvival to between 0.00001% and 0.1%. For all strains tested, theantimicrobial effect of L-lactate combined with Coumarin was greaterthan either of the compounds tested individually. This strongly suggeststhat L-lactate and Coumarin at commercially-applicable concentrationsexert a synergistic antimicrobial effect on E. coli strains.

The viability of various bacterial species in commercially applicableconcentrations of L-lactate and Coumarin was examined. Coumarin (6.8 mM)had no effect on the viability of the E. coli O157:H7 strain NCTC 12900(FIG. 11A) whereas L-lactate (2%) caused a decrease in viability ofapproximately 3 log₁₀ units in 10 minutes. Together, L-lactate andCoumarin decreased the viability of this E. coli O157:H7 strain bygreater than 7 log₁₀ units in 10 minutes. As shown in FIG. 11B, Coumarin(6.8 mM) alone had no effect on the viability of S. enteritidis whereasL-lactate reduced bacterial numbers by greater than 7 log₁₀ units in 5minutes. The combination of Coumarin and L-lactate reduced viability bygreater than 7 log₁₀ units in 1.5 minutes. As shown in FIG. 11C,Coumarin (6.8 mM) had no effect on the viability of L. monocytogeneswhereas L-lactate on its own reduced viability by greater than 7 log₁₀units in 20 minutes. Together Coumarin and L-lactate reduced theviability of this pathogen by greater than 7 log₁₀ units in 15 minutes.Coumarin had no effect on the viability of S. aureus (FIG. 11D) whereasL-lactate alone reduced viability by approximately 2 log₁₀ units in 60minutes. In combination, Coumarin and L-lactate reduced viability ofthis organism by greater than 5 log₁₀ units in 60 minutes. This stronglysuggests that organic acids and Coumarin at commercially-applicableconcentrations exert a synergistic antimicrobial effect on all bacterialpathogens.

The effect of temperature on the efficacy of commercially-applicableconcentrations of L-lactate (2%) and Coumarin (6.8 mM) on variouspathogens was investigated. As shown in FIG. 12A, the viability of theE. coli O157 strain NCTC 12900 at 37° C. was reduced by greater than 7log₁₀ units in 10 minutes. At 20° C., a greater than 5 log₁₀ unitreduction in viability was achieved in 2 hours and at 5° C. a greaterthan 5 log₁₀ unit reduction was achieved in 8 hours. The viability of S.enteritidis (FIG. 12B) was reduced by greater than 7 log₁₀ units in 1.5minutes at 37° C. and by a similar extent in 7.5 minutes at 20° C. At 5°C., viability was reduced by approximately 5 log₁₀ units in 60 minutes.The viability of L. monocytogenes (FIG. 12C) was reduced by greater than7 log₁₀ units in 15 minutes at 37° C. The same extent of reduction wasachieved after 60 minutes at 20° C. and after 120 minutes at 5° C. Asshown in FIG. 12D, Coumarin and L-lactate reduced the viability of S.aureus by greater than 5 log₁₀ units in 1 hour at 37° C. At 20° C.,viability was reduced by 5 log₁₀ units in 8 hours whereas at 5° C.viability was reduced by approximately 2 log₁₀ units in 8 hours.

For certain applications it is desirable to determine the potential forcontaminating E. coli O157:H7 strains to increase in numbers. Toevaluate the effect of L-lactate and coumarins on growing E. coliO157:H7 cells, NCTC 12900 was prepared as for the above experiments. Theculture was then diluted into fresh media and incubated at 37° C. for 2hours. L-lactate and/or a coumarin were added and the cultures werere-incubated over a period of time. Bacterial growth was monitored byspectrophotometer (650 nm) and compared to a control lacking bothL-lactate and a coumarin.

As illustrated in FIG. 13A, 12.5 mM L-lactate or 1.25 mM esculetinindividually caused a small reduction in the growth of the E. coliO157:H7 strain NCTC 12900 compared to the control. L-Lactate andesculetin added to the culture together had a synergistic effect, almostentirely retarding the growth of this strain. Compared to the control,0.625 mM of Coumarin only slightly reduced the extent of growth of NCTC12900 (FIG. 13B). Growth of this strain was inhibited substantially by18 mM L-lactate and the combination of Coumarin and L-lactate caused aneven greater reduction in growth. As shown in FIG. 13C, Coumarin (2 mM)inhibited the growth of S. enteritidis slightly and L-lactate inhibitedgrowth to a greater extent. Together, these compounds reduced the growthof S. enteritidis more than either compound on its own. As shown in FIG.13D, 1.25 mM Coumarin did not affect the growth of L. monocytogenes andL-lactate inhibited growth slightly. Together Coumarin and L-lactatereduced the growth of this organism substantially. The growth of S.aureus (FIG. 13E) was inhibited by Coumarin (1.25 mM) on its own and byL-lactate (5 mM) on its own. Together these compounds caused asynergistic decrease in the growth of this species. These resultsstrongly suggest that organic acids and coumarins synergisticallyinhibit growth of all bacterial pathogens.

REFERENCES

Bintsis et al. (2000) Food Microbiol. 17, 687-695.

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1. An anti-bacterial composition comprising an admixture of an organicacid and a coumarin or coumarin glycoside, wherein the organic acid isother than a short chain fatty acid.
 2. The composition as claimed inclaim 1 wherein the organic acid is heptanoic acid, decanoic acid,dodescanoic acid, sorbic acid, lactic acid, citric acid, benzoic acid,salicylic acid or succinic acid.
 3. The composition as claimed in claim2 wherein the organic acid is lactate, citrate or benzoic acid.
 4. Thecomposition as claimed claim 1 comprising 1 to 500 mM of the organicacid.
 5. The composition as claimed in claim 4 comprising 20 mM to 250mM of the organic acid.
 6. The composition as claimed in claim 5comprising 20 mM to 250 mM of L-lactate.
 7. The composition as claimedclaim 1 wherein the coumarin or coumarin glycoside is esculetin,scopoletin, umbelliferone, Coumarin, esculin or mixtures thereof.
 8. Thecomposition as claimed in claim 7 wherein the coumarin or coumaringlycoside is Coumarin.
 9. The composition as claimed in claim 1comprising 0.05 mM to 15 mM of a coumarin or coumarin glycoside.
 10. Thecomposition as claimed in claim 1 comprising at least 0.5 mM of acoumarin or coumarin glycoside.
 11. A method for reducing the infectiveability or for inactivating bacterial pathogens by contacting saidbacteria with a composition as claimed claim
 1. 12. The method asclaimed in claim 11 for use in food preparation.
 13. The method asclaimed in claim 11 for use in treatment of animals or humans infectedwith said pathogens.
 14. The method as claimed in claim 11 for use inthe disinfection of buildings or medical instruments.
 15. The method asclaimed in claim 11 wherein said bacterial pathogen is E. coli, Shigellaspp., Salmonella spp., Listeria spp., or Straphylococcus spp.
 16. Themethod as claimed in claim 15 wherein said bacterial pathogen is E. coli0157.
 17. The method as claimed in claim 15 wherein said bacterialpathogen is MRSA.
 18. (canceled)